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The neurotrophic factor receptor RET drives haematopoietic stem cell survival and function

Nature volume 514, pages 98–101 (2014)Cite this article

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Abstract

Haematopoiesis is a developmental cascade that generates all blood cell lineages in health and disease. This process relies on quiescent haematopoietic stem cells capable of differentiating, self renewing and expanding upon physiological demand1,2. However, the mechanisms that regulate haematopoietic stem cell homeostasis and function remain largely unknown. Here we show that the neurotrophic factor receptor RET (rearranged during transfection) drives haematopoietic stem cell survival, expansion and function. We find that haematopoietic stem cells express RET and that its neurotrophic factor partners are produced in the haematopoietic stem cell environment. Ablation of Ret leads to impaired survival and reduced numbers of haematopoietic stem cells with normal differentiation potential, but loss of cell-autonomous stress response and reconstitution potential. Strikingly, RET signals provide haematopoietic stem cells with critical Bcl2 and Bcl2l1 surviving cues, downstream of p38 mitogen-activated protein (MAP) kinase and cyclic-AMP-response element binding protein (CREB) activation. Accordingly, enforced expression of RET downstream targets, Bcl2 or Bcl2l1, is sufficient to restore the activity of Ret null progenitors in vivo. Activation of RET results in improved haematopoietic stem cell survival, expansion and in vivo transplantation efficiency. Remarkably, human cord-blood progenitor expansion and transplantation is also improved by neurotrophic factors, opening the way for exploration of RET agonists in human haematopoietic stem cell transplantation. Our work shows that neurotrophic factors are novel components of the haematopoietic stem cell microenvironment, revealing that haematopoietic stem cells and neurons are regulated by similar signals.

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Figure 1: Ret deficiency leads to reduced HSCs with impaired transplantation potential.
Figure 2: RET cell-autonomous signals control haematopoietic stress responses.
Figure 3: RET induces Bcl2/Bcl2l1 downstream of p38/MAP and CREB activation.
Figure 4: RET activation promotes HSC expansion and transplantation efficiency.

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Acknowledgements

We thank I. Monteiro Grillo and the radiotherapy service at Hospital de Santa Maria; H. Ferreira and the service of obstetrics, gynaecology and reproductive medicine at the Hospital of Santa Maria; the Instituto de Medicina Molecular animal facility, flow cytometry unit, bioimaging unit and histology unit for technical assistance. We also thank all members of H.V.-F. laboratory for discussion. D.F.-P., S.A.-M., R.G.D. and A.R.M.A. were supported by scholarships from Fundação para a Ciência e Tecnologia, Portugal. H.V.-F. was supported by Fundação para a Ciência e Tecnologia (PTDC/SAU-MII/104931/2008), Portugal, the European Molecular Biology Organisation (Project 1648), European Research Council (Project 207057) and National Blood Foundation, USA.

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Author notes

  1. Diogo Fonseca-Pereira and Sílvia Arroz-Madeira: These authors contributed equally to this work.

Authors and Affiliations

  1. Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Avenida Professor Egas Moniz, Edifício Egas Moniz, 1649-028 Lisboa, Portugal,

    Diogo Fonseca-Pereira, Sílvia Arroz-Madeira, Mariana Rodrigues-Campos, Inês A. M. Barbosa, Rita G. Domingues, Teresa Bento, Afonso R. M. Almeida, Hélder Ribeiro & Henrique Veiga-Fernandes

  2. Division of Molecular Immunology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK,

    Alexandre J. Potocnik

  3. Institute of Immunology and Infection Research, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK,

    Alexandre J. Potocnik

  4. Laboratory for Neuronal Differentiation and Regeneration, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan,

    Hideki Enomoto

  5. Graduate School of Medicine, Kobe University7-5-1 Kusunoki-cho, Chuo-ku, Kobe City, Hyogo 650-0017, Japan,

    Hideki Enomoto

Authors
  1. Diogo Fonseca-Pereira
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Contributions

D.F.-P., S.A.-M., M.R.-C., I.B., R.G.D., T.B., A.R.M.A. and H.R. did experiments and data analysis; H.E. generated RetBCLxL mice; D.F.-P., S.A.-M., A.P. and H.V.-F. designed in vivo and ex vivo experiments; D.F.-P. and H.V.-F. wrote the manuscript and H.V.-F. directed the study.

Corresponding author

Correspondence to Henrique Veiga-Fernandes.

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Extended data figures and tables

Extended Data Figure 1 Ret expression in haematopoietic progenitors and Ret ligand expression in the fetal and adult HSC environment.

a, FACS-sorted E14.5 fetal liver myeloid progenitors and LSK were analysed by RT–qPCR. b, FACS-sorted HSCs from E14.5 fetal liver and adult bone marrow were analysed by RT–qPCR. c, FACS-sorted E14.5 fetal liver and adult bone marrow HSCs were analysed by confocal microscopy. d, E14.5 fetal liver TER119−CD45−CD31+ endothelial cells (EC), TER119−CD45−CD31−cKit+ICAM-1− hepatocyte progenitor cells (HP) and TER119−CD45−CD31− cKit−ICAM-1+ mesenchymal cells (MC) were analysed by RT–qPCR (left); negative controls relative to Fig. 1c were analysed by confocal microscopy (right). e, Bone marrow TER119−CD45−CD31+Sca1+ endothelial cells (Endo), TER119−CD45−CD31−Sca1−CD51+ osteoblasts (Osteo) and TER119−CD45−CD31−Sca1+CD51+ mesenchymal stem cells (MSC) were analysed by RT–qPCR (left) and by confocal microscopy (right). f, E14.5 fetal liver and adult bone marrow were analysed by confocal microscopy. Arrows, candidate Lin−CD150+CD48−CD41− HSCs, relative to Fig. 1d, e. Figure shows negative controls for GFL staining. g, E15.5 gut tissue was analysed by confocal microscopy. White bar, 5 μm. Error bars, s.e.m. Housekeeping genes: Gapdh and Hprt1. ***P value for one-way ANOVA lower than 0.001.

Extended Data Figure 2 LSKs are affected by Ret deficiency and have reduced reconstitution capacity.

a, E14.5 total fetal liver cells. WT n = 20; Ret−/− n = 18. b, Flow cytometry analysis of E14.5 Ret−/− and WT littermate control LSKs (top) and myeloid progenitors (bottom). c, Flow cytometry analysis of Annexin V+ cells in cultured LSK cells. d, Flow cytometry analysis of E14.5 Ret−/− and WT littermate control HSCs. e, Flow cytometry analysis of E14.5 Ret−/− and WT Lin−CD150+CD48− HSC cells. Mean fluorescence intensity of cKit was analysed. No statistically significant differences were found. f, Lin−cKit+ cells were labelled with CMTMR. Percentage of Lin−cKit+CMTMR+ cells in bone marrow and spleen 20 h after injection (n = 3). g, h, Percentage of donor CD45.2 cells in blood cell lineages 16 weeks after primary and secondary (red) transplantation, relative to Fig. 2b, d. i, Day 12 CFU-s. WT n = 10; Ret−/− n = 10. Error bars, s.e.m. *, ** and ***, P values for Student’s t-test lower than 0.05, 0.01 and 0.001 respectively.

Extended Data Figure 3 Gfra1-deficient embryos have normal LSK numbers and reconstitution potential.

a, Flow cytometry analysis of E14.5 Gfra1−/− and WT littermate control LSKs (top) and myeloid progenitors (bottom). b, Number of LSKs and total fetal liver cells. WT n = 9; Gfra1−/− n = 10. c, Gfra1−/− or WT cells were injected with a third-party CD45.1/2 competitor. Percentage of donor CD45.2 cells in blood 16 weeks after transplantation. WT n = 5; Gfra1−/− n = 3. d, Flow cytometry analysis of bone marrow LSK cells 16 weeks after transplantation. Error bars, s.e.m.

Extended Data Figure 4 Ret conditional knockout mice and analysis of haematopoietic stem cells.

a, Ret conditional knockout. Scheme of the targeted Ret allele. b, E14.5 fetal liver HSCs from Retfl littermate controls (two animals) and Ret conditional knockout Vav1-iCre.RetΔ (three animals) and deleted bone marrow control (last column) were purified by flow cytometry. Efficient deletion of Ret in Vav1-iCre.RetΔ cells was determined by qPCR as fold increase relative to littermate control cells. c, Number of E14.5 LSKs and total fetal liver cells. Retfl n = 8; RetΔ n = 8. d, Scheme of competitive transplantation with RetΔ animals and littermate controls, relative to Fig. 2h. e, Percentage of donor CD45.2 cells in blood cell lineages 16 weeks after primary and secondary transplantation. Error bars, s.e.m. *, ** and ***, P values for Student’s t-test lower than 0.05, 0.01 and 0.001 respectively.

Extended Data Figure 5 Ret expression increases after haematopoietic stress, and RET signalling increases CREB phosphorylation and cell survival.

a, Mice were sublethally irradiated. Irradiation-induced stressed HSCs were purified by flow cytometry at 72 h and analysed by RT–qPCR. b, RT–qPCR for fetal liver E14.5 Ret−/− and WT HSCs (n = 3). c, Confocal analysis of BCLxL expression in WT and Ret−/− HSCs. d, Flow cytometry analysis of Annexin V+ cells in cultured LSK cells. e, Flow cytometry of E14.5 Ret−/− and WT littermate controls. P-Akt (T308), P-S6 and PIP3: WT n = 6, Ret−/− n = 6. f, Flow cytometry analysis of LSK cells in the absence or presence of GDNF, NRTN or ARTN for 1 h (n = 6). g, Bcl2 expression in LSK cells upon GFL treatment and with different inhibitors, relative to LSKs treated with GFLs only. Light grey, isotype control. White bar, 5 μm. Error bars, s.e.m. * and **, P values for Student’s t-test lower than 0.05 and 0.01 respectively.

Extended Data Figure 6 Rescue of haematopoietic progenitors with Ret and its downstream targets.

a, Scheme of competitive transplantation, relative to Fig. 4b, c. b, Flow cytometry analysis of donor CD45.2 blood cells at 8 weeks upon transplantation of Bcl2l1-transduced WT haematopoietic progenitor cells. Error bars, s.e.m.

Extended Data Figure 7 Generation and analysis of RetBCLxL mice.

a, Ret locus was targeted by a construct containing the BCLxL coding sequence, an internal ribosomal entry site (IRES) and a puromycin resistance cassette, followed by a floxed neomycin resistance cassette to aid negative selection. Ret Bcl-xL.IRES.Puromicin knock-in mice were obtained by excising of the neomycin cassette. b, Number of myeloid progenitors, LSK cells, multipotent progenitor cells and HSCs in E14.5 fetal liver. WT n = 7; Ret+/BCLxL n = 9; RetBCLxL/BCLxL n = 8. c, FACS-sorted HSCs from Ret+/+, E14.5 fetal liver Ret+/BCLxL and adult bone marrow Ret+/BCLxL were analysed by RT–qPCR for human BCL2L1 expression. Housekeeping genes: Gapdh and Hprt1. d, Annexin V+ cells in cultured E14.5 LSK cells. WT n = 6; Ret+/BCLxL n = 4; RetBCLxL/BCLxL n = 6. e, Scheme of competitive transplantation. RetBCLxL/BCLxL animals and littermate controls were injected in competition with CD45.1/CD45.2 cells, relative to Fig. 4d, e. Error bars, s.e.m. ***P value for one-way ANOVA lower than 0.001.

Extended Data Figure 8 GFLs increase anti-apoptotic gene expression in human haematopoietic progenitors.

Human cord blood CD34+ cells were cultured in the presence or absence of GFLs for 4 days and analysed by RT–qPCR. Gene expression relative to cells cultured without GFLs. Error bars, s.e.m. ***P values for Student’s t-test lower than 0.001.

Extended Data Figure 9 Gfra2- and Gfra3-deficient embryos have normal haematopoietic progenitors.

a, Flow cytometry analysis of E14.5 Gfra2−/− and WT littermate control LSKs (top) and myeloid progenitors (bottom). b, Number of LSKs and total fetal liver cells. WT n = 12; Gfra2−/− n = 11. c, Flow cytometry analysis of E14.5 Gfra3−/− and WT littermate control LSKs (top) and myeloid progenitors (bottom). d, Number of LSKs and total fetal liver cells. WT n = 11; Gfra3−/− n = 20. Error bars, s.e.m.

Extended Data Figure 10 Neuronal growth factors regulate HSC response to physiological demand.

The neurotrophic factors GDNF, NRTN and ARTN are produced by cells in the HSC microenvironment and act directly on HSCs by activation of RET. Highlighted area: RET stimulation results in p38/MAP kinase and CREB activation leading to Bcl2 and Bcl2l1 expression. RET signals provide HSCs with survival signals that preserve HSC stemness.

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Fonseca-Pereira, D., Arroz-Madeira, S., Rodrigues-Campos, M. et al. The neurotrophic factor receptor RET drives haematopoietic stem cell survival and function. Nature 514, 98–101 (2014). https://doi.org/10.1038/nature13498

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